Yee et al. (J. Immunol. 162 (1999), 2227-2234) describe the isolation of high avidity melanoma-reactive cytotoxic T lymphocytes (CTL) from heterogeneous populations using peptide-MHC tetramers. The MHC tetramers are biotinylated and irreversibly conjugated to avidin containing a fluorescent labeling group. A removal of the fluorescent marker under physiological conditions is thus not possible. Youde et al. (Cancer Res. 60 (2000), 365-371) describe the use of fluorescence labelled MHC tetramers to isolate human CTLs recognizing endogeneous human papilloma virus antigens. Also here biotinylated MHC molecules are used, which are irreversibly bound to streptavidin under physiological conditions so that a removal of the detection reagent from the T cells is not possible.
U.S. Pat. No. 5,985,658 describes a method for separating target cells from a plurality of cells which is based on a reversible high affinity interaction. The method comprises forming a target cell/cell binding reagent/first molecule/second molecule/solid support complex, wherein the cell binding reagent is specific for target cells, e.g. an antibody, wherein the first molecule reversibly binds to the second molecule, wherein one of the first and second molecules is calmodulin, and wherein the other of the first and second molecules is a calmodulin-binding peptide. After removal of non-target cells not attached to the solid support the binding between first and second molecule is reversed, thereby releasing target cells as separate cells from the plurality of starting cells.
The disadvantage of this method is that the target cell binding agent must have a high affinity for the target cell, in order to enable an isolation of the target cell population. Thus, after removal of the binding between first and second molecule the target cell-specific reagent remains bound to the target cell.
Werther et al. (J. Immunol. Meth. 238 (2000), 133-141) describe an antibody-based protocol for the isolation of carcinoma cells from mononuclear cell suspensions. The target cells are stained with an antibody linked to a magnetic bead and thus separated from the starting cell population. The magnetic bead is bound to the target cell via a DNA linker which may be digested with DNase in order to allow a further phenotypical characterization of the target cells. Also this method has the disadvantage that a high affinity target cell binding reagent, i.e. an antibody has to be used, which cannot be easily removed from the target cell.
Marelli-Berg et al. (J. Immunol. 244 (2000), 205-215) describe a protocol for the isolation of endothelial cells from murine tissue. Endothelial cells are labelled by specific antibodies and then purified via binding to microbeads. Also this method includes the above mentioned disadvantages.